Revista da Sociedade Brasileira de Medicina Tropical

Journal of the Brazilian Society of Tropical Medicine

Vol.:53:(e20200451): 2020
https://doi.org/10.1590/0037-8682-0451-2020

Mini Review

Laboratory diagnosis for Covid-19: A mini-review

Juliana Lemos Dal Pizzol[1], Vanusa Pousada da Hora[1], Ana Júlia Reis[1],
Júlia Vianna[1], Ivy Ramis[1], Andrea von Groll[1] and Pedro Almeida da Silva[1]

[1]. Universidade Federal do Rio Grande, Núcleo de Pesquisa em Microbiologia Médica,

Programa de Pós-Graduação em Ciências da Saúde, Rio Grande, RS, Brasil.

Abstract
Coronavirus disease (COVID-19) is a pandemic caused by a new coronavirus, called SARS-CoV-2. This disease was first identified in
December 2019 and rapidly developed into a challenge to the public health systems around the world. In the absence of a vaccine and
specific therapies, disease control and promotion of patient health are strongly dependent on a rapid and accurate diagnosis. This review
describes the main laboratory approaches to making a diagnosis of COVID-19 and identifying those previously infected with SARS-CoV-2.
Keywords: COVID-19. Diagnostic. Serology. Molecular. Biomarkers.

INTRODUCTION SEROLOGICAL TESTS

Rapid and accurate diagnosis of Coronavirus disease
(COVID-19) is essential for pandemic control as well as for
establishing an adequate therapeutic strategy to reduce morbidity
and mortality. For both epidemiological and clinical purposes,
several methodological approaches have been developed. In this
article, we will cover the main laboratory methods and protocols
that have been used for the control and management of COVID-19.
USING LABORATORY DIAGNOSIS TO ENHANCE THE
CONTROL OF COVID-19
Reliable laboratory diagnosis represents one of the main tools
for the promotion, prevention, and control of infectious diseases1.
The diagnostic methods for COVID-19 fall under two main
categories: immunological and molecular. Immunological tests can
be serological tests that mainly detect antibodies in blood or viral
antigens in respiratory secretions, and both can be performed with
point-of-care platforms. Regarding molecular tests, they are based
on the detection of SARS-CoV-2 RNA mainly in nasopharyngeal
samples, which in most cases require adequate laboratory
infrastructure. In addition to the cited tests, other laboratory
parameters have been used as an aid in the clinical monitoring of
patients with COVID-192-4.
Corresponding author: Prof. Pedro Almeida da Silva.
e-mail: pedrefurg@gmail.com
 https://orcid.org/0000-0003-1666-1295
Received 2 July 2020
Accepted 5 August 2020
Serological tests are especially important for the diagnosis
of patients with mild to moderate disease, in the absence of
molecular diagnostics5. These tests can have several benefits, such
as estimating the transmissibility and lethality rates, assessing
individual and community immunity, and valuing the need and
effectiveness of nonpharmaceutical interventions (e.g., social
isolation). Furthermore, the plasma of convalescents with high
levels of antibody production could be used as a therapeutic support6.
Several serological tests based on enzyme-linked immunosorbent
assay (ELISA), and lateral flow immunochromatography (LFI)
devices have been developed by different companies worldwide.
IgM and IgG antibodies detected on ELISA have more than 95%
specificity in the diagnosis of COVID-19 (18). High titers of IgG
antibodies detected by ELISA demonstrate a positive correlation
with neutralizing antibodies7.
Given their point-of-care characteristics, LFI platforms
have been widely used. In general, this method detects IgM and
IgG antibodies in approximately 20 minutes, individually or
simultaneously. Antibodies to glycoprotein S (spike) are analyzed
from blood samples obtained by finger puncture without the need
for sophisticated equipment or specialized professionals8. However,
these tests are purely qualitative and can only indicate the presence
or absence of SARS-CoV-2 antibodies5. Despite its potential value
as a tool for pandemic control, the validation of LFI tests remains
challenging9. The ability to assess their accuracy (sensitivity and
specificity) as well as their ability to monitor immunity over time
remains insufficient10.

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Dal Pizzol JL et al. - Laboratory diagnosis for COVID-19
Another matter of concern is inappropriate interpretation of the
result, such as a false understanding that a positive result indicates
immunity against the SARS-CoV-2, whereas a positive result on
the serological test indicates that the person has come into contact
with the virus and developed antibodies, but it is not clear whether
these antibodies will provide protection against a reinfection11.
Currently, antibody responses against SARS-CoV-2 remain
poorly understood, and the clinical usefulness of the serological test
is still unclear12. Although the detection of IgM and IgG by ELISA
is positive even on the fourth day after the onset of symptoms, high
levels of these antibodies are produced in the second and third
weeks of the disease5. From the time of onset, the IgM antibody
titer increases; 2 weeks after the onset of symptoms, both IgG and
IgM are present and their levels start to decrease after the fourth
week. IgM is notoriously nonspecific, and because it takes weeks to
develop specific IgG responses, serological detection is unlikely to
play an active role in case management, with diagnosis/confirmation
of late cases of COVID-19 or determining the immunity of health
professionals being the exceptions12. The acute antibody response
to SARS-CoV-2 in 285 patients in China’s Hubei province was
detected using a chemiluminescence immunoenzymatic test
(CLIA). The result showed that the proportion of patients positive
for specific IgG reached 100% approximately 17 to 19 days after
the onset of symptoms. Meanwhile, the proportion of patients with
specific IgM reached 94.1% at 20 to 22 days after the onset of
symptoms. Seroconversion to IgG and IgM occurred simultaneously
or sequentially, giving an average seroconversion time of 13 days
after the onset of symptoms. The study data indicate that serological
tests can be complementary, especially in the diagnosis of suspected
patients with negative molecular results and also in the search for
asymptomatic infections among close contacts13.
An interesting aspect is that the most severe cases have higher
levels of IgM and IgG in than the mild cases14,15. In this context,
the quantitative detection of antibodies can be an important aid in
clinical practice16.
It is worth mentioning that the majority of immunoassays for
SARS-CoV-2 have immunogenic proteins as their main target: 1)
protein S (spike), which is the most highly exposed viral protein, and
2) nucleocapsid protein (N), which is abundantly expressed during
TABLE 1: List of RT-PCR protocols indicated by the WHO.
Institute
China CDC, China
Institute Pasteur, France
US CDC, USA
National Institute of Infectious Diseases, Japan
Charité, Germany
HKU, Hong Kong SAR
National Institute of Health, Thailand
(Source: https://www.who.int/who-documents-detail/molecular-assays-to-diagnose-COVID-19-summary-table-of-available-protocols)
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infection17. Normally, most antibodies are produced against the most
abundant protein present in the virus (N). Therefore, tests that detect
antibodies to N would be the most sensitive. However, the protein S
receptor binding domain (RBD-S) is the host's attachment protein,
and antibodies to RBD-S would be very specific and are expected
to be neutralizing. In this case, the use of one or both antigens to
detect IgG and IgM would result in high sensitivity5,7.
MOLECULAR TESTS
Most molecular tests, unlike serological tests, are performed
in a specialized laboratory using cutting-edge equipment and
highly qualified staff, so their use is limited. Nasopharyngeal (NP)
swabs are considered the standard samples for the detection of
SARS-CoV-2. In addition to the NP swabs, the use of samples
from the lower respiratory tract (sputum or bronchial lavage) and
oropharyngeal (OP) swabs are used as alternatives to improve the
biosafety of health care workers18. A robust study on the detection of
SARS-CoV-2 in different clinical samples involved 205 hospitalized
patients from three Chinese hospitals, with a total of 1,070 samples
collected. The NP swabs had the highest viral load, although at the
time of collection, the stage of disease or the associated clinical
history was not available for many of these patients2,19. For the
detection of SARS-CoV-2 by this technique, samples must be
collected when the patient is in the acute phase of infection,
preferably up to 5 days after the onset of symptoms20. This method
has the advantage of being both quantitative and highly specific18.
Reverse transcription followed by real-time reverse transcription
polymerase chain reaction (RT-PCR) is considered the gold standard
for the diagnosis of COVID-19. The first protocol recommended by the
WHO was published by Charité Institute, Berlin University, Germany21.
It is based on TaqMan technology, with indicated primers and probes
to detect the RNA-dependent RNA polymerase (RdRp), envelope
protein (E), and nucleocapsid protein (N) genes. Subsequently,
several in-house methods have been reported by the WHO, and
they are being validated in WHO partner laboratories (Table 1).
False-negative results can occur mainly because of inadequate
extraction of nucleic acid; poor sample quality; low viral load;
sample collection time; incorrect sample storage, transportation,
and handling; and PCR inhibition22-24.
Gene targets
ORF1ab and N
Two targets in RdRP
Three targets in N gene
Pancorona and multiple targets, Spike protein
RdRP, E, N
ORF1b-nsp14, N
N

Results of different RT-PCRs protocols have shown variation
in their performance depending on the primers and probes. One
comparative study of the sensitivity of SARS-CoV-2 RT-PCR
tests developed by Charité (Germany), HKU (Hong-Kong), China
CDC (China), US CDC (United-States), and Institut Pasteur,
Paris (France) showed that all RT-PCR assays performed well for
SARS-CoV-2 detection, but the authors pointed out that the assays
of RdRp Institut Pasteur (IP2, IP4), N China CDC, and N1 US CDC
were the most sensitive25.
The use of specific primers determines the high specificity of
RT-PCR, and the possibility of false-positives cannot be excluded.
In this sense, a negative template control should be introduced in
every RT-PCR26. A chest computerized tomography (CT) scan can
be used as a complementary diagnostic tool that enables physicians
to effectively detect COVID-19 infection in several RT-PCR
false-negative cases. Repeat tests can be particularly important
if the patient has a clinical picture of viral pneumonia, history of
exposure, and/or radiographic findings (CT or magnetic resonance
imaging) compatible with COVID-19 pneumonia12.
The development of new diagnostic platforms can improve
molecular methods in terms of speed, sensitivity, and accessibility
in the diagnosis of COVID-19. Currently, approximately 11
molecular devices have received urgent approval from the National
Administration of Medical Products in China18. In addition,
automated RT-PCR systems have been developed, such as the
Xpress SARS CoV-2 Test (Cepheid, USA). Using the GeneXpert
platform, SARS-CoV-2 E and N2 genes can be detected in
approximately 45 minutes. Another innovative alternative is the
Abbott ID Now COVID-19 handheld instrument, which detects the
SARS-CoV-2 RdRp and N genes. Both Xpress SARS-CoV-2 and
Abbott ID Now COVID-19 were authorized for emergency use by
the Food and Drug Administration (FDA), USA27,28.
However, with the global shortage of kits, many countries have
begun to carry out in-house RT-PCR to overcome this shortage.
The most frequently used in-house protocols are as follows: 1)
Hospital Charité - University of Berlin that targets the genes E, N
and RdRp21, endorsed by the WHO, 2) CDC-China, which targets
the ORF1ab and N genes (CDC-CHINA 2020), and 3) CDC-USA,
which uses three targets within the N gene29.
Another molecular approach that may be useful, especially
in places where there is no need for expensive thermocycling
equipment is reverse transcription loop-mediated isothermal
amplification (RT-LAMP) using the SARS-CoV-2 spike ORF1ab
and S genes30,31. In addition, the use of biosensors to detect SARS-
CoV-2 viral RNA has also been tested. The newly developed
biosensor integrates the plasmatic photothermal effect and
plasmon resonance detection transduction. Validity and selectivity
were determined by using the SARS-CoV-2 RdRp and ORF1ab
sequences as targets32.
BIOSAFETY
Laboratory testing is a process divided into three main phases:
preanalytical, analytical, and postanalytical. In the preanalytical
stage, individual protection measures in relation to sample collection
are fundamental to good biosafety practices. Based on the WHO
Rev Soc Bras Med Trop | on line | Vol.:53:(e2020451): 2020
guidelines, all clinical samples should be considered potentially
infectious, and health professionals must use suitable and a complete
set of personal protective equipment (PPE) when obtaining/handling
patient samples. Normally, the use of a disposable apron, gloves,
bonnet, foot protection, protective goggles, and an N95 breathing
mask is expected.
Clinical samples must be transported to the laboratory in
packaging appropriate for level-2 biological risk inside a leak-proof
cryogenic box and with a clearly visible biohazard label33. The
samples can be stored at 2–8°C for up to 72 hours after collection.
If the sample needs to be stored for long, it must be done at a
temperature of at least -70°C. The extracted nucleic acid must also
be stored at the same minimum temperature of -70°C29.
Sample processing must be done inside a class-2 biological
cabin using suitable clothing and a complete set of PPE. All
surfaces, cryo-boxes, and equipment must be cleaned using 0.1%
sodium hypochlorite, 62-71% ethanol, 0.5% hydrogen peroxide,
and quaternary ammonium or phenolic compounds34.
NON-SPECIFIC LABORATORY TESTS
Results of laboratory tests can increase the support for the
diagnosis, prognosis, and monitoring of patients through the
detection and measurement of different biomarkers. Although
nonspecific, some biomarkers have been reported to be associated
with the infectious process of SARS-CoV-2. Therefore, low
lymphocyte and platelet counts; low serum albumin levels; and
increased serum levels of C-reactive protein, D-dimer, ferritin,
lactate dehydrogenase, transaminases, and interleukin-6 can be
used in risk stratification to predict the severity of COVID-1935-39.
In addition, cytokine storms with high levels of IL-2R, IL-6, IL-10,
and TNF-α and a reduction in the absolute numbers of CD4 + and
CD8 + T lymphocytes have been related to severe cases of
COVID-1940, with progression to cardiovascular collapse, multiple
organ failure, and rapid deaths41.
DISCUSSION
This review provides an overview of the diagnostic methods
used for COVID-19. New studies on this topic are rapidly becoming
available in the literature, but there are crucial gaps that prevent an
effective response in this pandemic. Although RT-PCR is the most
widely used option for diagnosis, it can provide false-negative
results, and its use is limited by the requirement of laboratory
infrastructure and high costs22,42,43. Molecular point-of-care tests,
with high precision, that present fast results, low cost, and easy
execution are urgently needed to expand the diagnostic capacity
of health systems, especially in low-income settings.
Serological tests can function as complementary tools for the
diagnosis of COVID-19. Despite the fact that positive serological
results can be obtained around 4–7 days after the onset of the
symptoms, their usefulness for patients with viral loads below
the detection limit of real-time RT-PCR assays is questionable19.
These tests are also important to understand the epidemiology of
SARS-CoV-2, including the role of asymptomatic infection and
current or past infection44.
Although diagnosis by real-time RT-PCR is still essential
to identify acute SARS-CoV-2 infection, serological tests and
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Dal Pizzol JL et al. - Laboratory diagnosis for COVID-19
epidemiological data are necessary to understand past pandemics
and predict their future9,10. The Global Health Security (GHS) index
shows that only 19% of the countries have the capacity to detect
and report epidemics of potential international concern, with fewer
than 5% of the countries having the ability to respond quickly and
mitigate the spread of an epidemic. In this sense, it is clear that no
country has shown total preparedness for epidemics or pandemics45.
The global spread of COVID-19 should help nations establish
new and fundamental priorities in the field of research and public
health policies. The costs of lives lost and economic crises should
serve as stimuli, especially for emerging economies, so that
industrial-scale production in the health sector is accelerated and
able to meet national requirements.
ACKNOWLEDGMENTS
We express our deepest thanks to the Universidade Federal do
Rio Grande – FURG for supporting the development of this study.
AUTHORS’ CONTRIBUTION
JLDP: Acquisition of data, drafting the article, final approval
of the version to be submitted. VPH, AJR, JV and IR: Data
acquisition, analysis, and interpretation; final approval of the version
to be submitted. AVG: Data acquisition, analysis, and interpretation;
final approval of the version to be submitted. PAS: Conception and
design of the study, data acquisition, analysis, and interpretation;
drafting the article; final approval of the version to be submitted.
FINANCIAL SUPPORT
CAPES, CNPq, NIH, and FAPERGS.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
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